U.S. patent number 8,629,317 [Application Number 10/577,061] was granted by the patent office on 2014-01-14 for non-human transgenic mammal for the constant region of the class a human immunoglobulin heavy chain and applications thereof.
This patent grant is currently assigned to Centre National de la Recherche Scientifique, Universite de Limoges. The grantee listed for this patent is Micael Bardel, Michel Cogne, Catherine Decourt, Caroline Le Morvan, Christophe Sirac. Invention is credited to Micael Bardel, Michel Cogne, Catherine Decourt, Caroline Le Morvan, Christophe Sirac.
United States Patent |
8,629,317 |
Cogne , et al. |
January 14, 2014 |
Non-human transgenic mammal for the constant region of the class a
human immunoglobulin heavy chain and applications thereof
Abstract
The invention relates to a non-human transgenic mammal with an
IgH locus modified by replacement of the switching sequence S.mu.
with all or part of a transgene comprising the gene C.alpha. of a
class A human immunoglobulin, including at least the exon, coding
for the CH3 domain and the membrane exon and the applications of
the above for the production of humanized class IgA antibodies.
Inventors: |
Cogne; Michel (Isle,
FR), Sirac; Christophe (Limoges, FR),
Bardel; Micael (Couzeix, FR), Decourt; Catherine
(Rilhac-Rancon, FR), Le Morvan; Caroline
(Vicq-sur-Breuil, FR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Cogne; Michel
Sirac; Christophe
Bardel; Micael
Decourt; Catherine
Le Morvan; Caroline |
Isle
Limoges
Couzeix
Rilhac-Rancon
Vicq-sur-Breuil |
N/A
N/A
N/A
N/A
N/A |
FR
FR
FR
FR
FR |
|
|
Assignee: |
Centre National de la Recherche
Scientifique (Paris Cedex, FR)
Universite de Limoges (Limoges Cedex, FR)
|
Family
ID: |
34400790 |
Appl.
No.: |
10/577,061 |
Filed: |
October 21, 2004 |
PCT
Filed: |
October 21, 2004 |
PCT No.: |
PCT/FR2004/002701 |
371(c)(1),(2),(4) Date: |
February 27, 2007 |
PCT
Pub. No.: |
WO2005/047333 |
PCT
Pub. Date: |
May 26, 2005 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20070248601 A1 |
Oct 25, 2007 |
|
Foreign Application Priority Data
|
|
|
|
|
Oct 24, 2003 [FR] |
|
|
03 12502 |
|
Current U.S.
Class: |
800/13; 800/4;
435/320.1; 424/93.21; 800/8; 800/21 |
Current CPC
Class: |
A01K
67/0278 (20130101); A61P 35/00 (20180101); A61P
31/00 (20180101); C12N 15/8509 (20130101); C07K
16/18 (20130101); A01K 2267/01 (20130101); C07K
2317/21 (20130101); A01K 2227/105 (20130101); C12N
2800/30 (20130101); A01K 2217/00 (20130101); A01K
2207/15 (20130101); C07K 2317/24 (20130101) |
Current International
Class: |
A01K
67/027 (20060101); C12N 15/74 (20060101); C12P
21/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Qiu et al. Intl Immunol 1999;11:37-46. cited by examiner .
GeneBank AC073553 (Sep. 2002). cited by examiner .
Moreadith et al., J. Mol. Med. 1997;75(3):208-16. cited by examiner
.
Mullins et al. Journal of Clinical Investigation, 1996. cited by
examiner .
Denning, Nat Biotech 2001;19:559-562. cited by examiner .
Yanagimachi, Mol Cell Endocrinol 2002;187:241-8. cited by examiner
.
Wilmut, Cloning Stem Cell 2003;5:99-100. cited by examiner .
Polejaeva et al, Nature 2000;407:86. cited by examiner .
Harriman et al. J Clin Invest 1996;97-477-85. cited by examiner
.
Luby et al. J Exp Med 2001;193:159-68. cited by examiner .
Wall, Cloning Stem Cells 2001;3:209-220. cited by examiner .
Scinicariello et al. Immunol 2001;103:441-8. cited by examiner
.
XP-011182522 "Localization of the 3' IgH Locus Elements that Effect
Long-Distance Regulation of Class Switch Recombination" by Eric
Pinaud et al., Immunity, vol. 15, 187-199, Aug. 2001. cited by
applicant .
XP-0011182373 Research Report "Production of Antigen-Specific Human
Monoclonal Antibodies: Comparison of Mice Carrying IgH/.kappa. or
IgH/.kappa./.lamda. Transloci" by S. Magadan et al., BioTechniques
vol. 33, No. 3 pp. 680-690, Sep. 2002. cited by applicant .
"Insertion of the IgH locus 3' regulatory palindrome in expression
sectors warrants sure and efficient expression in stable B cell
transfectants" by Christine Chauveau et al., Gene 222 (1998)
279-285. cited by applicant .
"Use of a simple, general targeting vector for replacing the DNA of
the heavy chain constant region in mouse hybridoma cells" by Diana
Ronai et al., Journal of Immunological Methods 275 (2003) 191-202.
cited by applicant .
XP-002319762 "Immunoglobulin class-switch recombination in mice
devoid of any S.mu. tandem repeat" by Ahmend Amine Khamlichi et
al., Blood, May 15, 2004, vol. 103, No. 10, pp. 3828-3836. cited by
applicant.
|
Primary Examiner: Li; Janice
Attorney, Agent or Firm: Beaumont; William E.
Claims
The invention claimed is:
1. A transgenic mouse, wherein an endogenous IgH locus comprises
replacement of its switch sequence S.mu. with a transgene
consisting essentially of a human class A immunoglobulin heavy
chain constant region gene C.alpha. or a segment of said C.alpha.
gene comprising at least an exon encoding the CH3 domain and a
membrane exon, wherein said transgenic mouse produces chimeric
immunoglobulins A whose heavy chains comprise a mouse variable
region and a human constant region or a segment thereof comprising
at least the CH3 domain, and wherein said transgenic mouse produces
no mouse immunoglobulins M.
2. The transgenic mouse of claim 1, which is homozygous for said
modified IgH locus.
3. The transgenic mouse of claim 1, wherein said transgene consists
of the entire C.alpha. gene.
4. The transgenic mouse of claim 1, wherein said transgene consists
of the segment of the C.alpha. gene comprising the exon encoding
the CH3 domain and the membrane exon.
5. The transgenic mouse of claim 1, wherein said C.alpha. gene is
the C.alpha.1 gene.
6. The transgenic mouse of claim 1, which further comprises another
transgene encoding a human immunoglobulin light chain.
7. The transgenic mouse of claim 6, wherein said light chain is a
kappa light chain.
8. The transgenic mouse of claim 6, wherein said transgene which
encodes a human immunoglobulin kappa light chain, further comprises
the intronic activator E.mu. upstream of a DNA sequence encoding
said human immunoglobulin kappa light chain and the palindrome
hs3a/hs1,2/hs3b downstream of said DNA sequence.
9. The transgenic mouse of claim 8, wherein said transgene is under
the control of the promoter of the human immunoglobulin heavy
chain.
10. The transgenic mouse of claim 6, which is dizygous for said
transgene.
11. The transgenic mouse of claim 6, further comprising an
inactivated endogenous immunoglobulin kappa light chain locus.
12. The transgenic mouse of claim 11, which is homozygous for said
inactivated endogenous immunoglobulin kappa light chain locus.
13. The transgenic mouse of claim 1, further comprising an
inactivated endogenous J chain gene.
14. The transgenic mouse of claim 13, which is homozygous for said
inactivated endogenous J chain gene.
15. The transgenic mouse of claim 13, which further comprises
another transgene encoding a human immunoglobulin J chain gene.
16. The transgenic mouse of claim 1, wherein said: a) endogenous
mouse IgH locus comprises the replacement of its switch sequence
S.mu. with the entire human class A immunoglobulin heavy chain
constant region gene C.alpha.1, and b) which transgenic mouse
further comprises a human kappa light chain transgene comprising a
V.kappa.I gene rearranged with a J.kappa.5 gene, a
J.kappa.-C.kappa. intron and a C.kappa. gene, under the
transcriptional control of the human heavy chain promoter (pVH),
the intronic activator E.mu. upstream of said promoter of and the
palindrome hs3a/hs1,2/hs3b downstream of said C.kappa. gene.
17. A homologous recombination targeting vector, which comprises a
human class A immunoglobulin heavy chain constant region gene
C.alpha. or a segment of said C.alpha. gene comprising at least an
exon encoding the CH3 domain and a membrane exon, flanked by the
sequences SEQ ID NO: 7 and SEQ ID NO: 8.
18. The targeting vector of claim 17, which comprises a cassette
for expressing a selection marker, adjacent to said C.alpha. gene
or to a segment of said gene.
19. The targeting vector of claim 18, wherein said expression
cassette is flanked by site-specific recombination sequences.
20. The targeting vector of claim 19 wherein said sequences are
LoxP sequences of Cre recombinase.
21. A mouse embryonic cell, which is modified with the targeting
vector of claim 17.
22. A method for preparing humanized class IgA antibodies or
fragments thereof, which comprises at least the following steps: a)
immunizing the transgenic mouse of claim 1, and b) producing
humanized class IgA antibodies or fragments of the antibodies from
serum secretions or B lymphocytes of said transgenic mouse
sacrificed beforehand.
Description
RELATED APPLICATIONS
The present application is based on, and claims priority from
French Patent Application Number 0312502, filed Oct. 24, 2003, the
disclosure of which is hereby incorporated by reference herein in
its entirety.
The present invention relates to non-human mammal transgenic for
the constant region of the class A human immunoglobulin heavy chain
and to its applications for the production of humanized class IgA
antibodies.
The class A immunoglobulins (IgA) comprise two identical heavy
chains of isotype .alpha.1 (subclass IgA1) or .alpha.2 (subclass
IgA2) in humans, combined via disulfide bridges with two identical
light chains of isotype kappa (.kappa.) or lambda (.lamda.).
The .alpha. heavy chain, which is specific to this class of
immunoglobulins, exists in membrane form and in secreted form. The
secreted form comprises four domains of about 110 amino acids: a
variable domain VH and three constant domains CH1, CH2 and CH3, and
a hinge (H) region between CH2 and CH3 and a C-terminal
octapeptide. The penultimate cysteine of this octapeptide can form
a covalent bond with the J chain (or joining piece) which serves to
combine two IgA heavy chains so as to form dimeric IgAs. The
membrane form additionally comprises a hydrophobic domain allowing
anchoring of the protein in the membrane, and an intracytoplasmic
domain. The region of the heavy chain corresponding to the CH1,
CH2, H and CH3 domains combined either with the C-terminal
octapeptide (secreted form) or with the hydrophobic and
intracytoplasmic domain (membrane form) is called constant region
by contrast to the region corresponding to the variable domain VH
which is called variable region.
The .kappa. and .lamda. light chains, which are common to all the
classes and subclasses of immunoglobulins, comprise two domains: a
variable domain (VL) and a constant domain (CL). In humans, the
expression of the .kappa. and .lamda. chains is equivalent, whereas
in mice, the expression of the .lamda. locus is very low such that
95% of the light chains are of the .kappa. type. The region of the
light chain corresponding to the CL domain is called constant
region by contrast to the region corresponding to the variable
domain VL, which is called variable region.
The immunoglobulin genes are organized into loci, one locus for the
heavy chains (IgH locus) and one locus for each of the light chains
(lambda and kappa loci).
The loci of the light chains each comprise V and J genes encoding
the variable domain and C genes encoding the constant domain;
during the differentiation of the B lymphocytes, a V gene is
rearranged with a J gene and a C gene, and the V region is
additionally subjected to somatic mutations which make it possible
to produce antibodies with high affinity for the antigen.
The locus of the heavy chains comprises V, D and J genes encoding
the variable domain and C (C.mu., C.delta., C.gamma., C.epsilon.
and C.alpha.) genes encoding the constant domains of the isotypes
of the different classes of immunoglobulins; each C gene, except
C.delta., is preceded by a switch (S) sequence. The C.alpha.
(C.alpha.1 and C.alpha.2 in humans) genes contain introns
separating the exons encoding the constant domains CH1, CH2 and CH3
and the membrane (mb) exon; the sequence encoding the hinge region
is included in the exon cH2. During the differentiation of the B
lymphocytes, a V gene is rearranged with a D gene and a J gene, and
the V region is also subjected to somatic mutations which make it
possible to produce antibodies with high affinity for the antigen.
In addition, while the primary response to the antigen mainly
consists of IgM, the secondary response is associated with the
class switch mechanism during which the switch sequence S.mu.,
situated upstream of C.mu. recombines with another switch sequence,
thus leading to the production of another class of immunoglobulin
(IgG, IgE or IgA).
The diversity of the antibodies produced in response to the
stimulation by an antigen results from the combination of several
mechanisms: the multiplicity of the V genes, the somatic mutation
of these V genes, the somatic recombination of the V genes and the
somatic recombination of the switch sequences.
The IgAs exist in the body in two different forms: a serum IgA and
a secretory IgA (s-IgA).
The serum IgA represents 15 to 20% of the serum immunoglobulins;
more than 80% of the human serum IgA is in monomeric form, whereas
in most other mammalian species it is essentially in dimeric
form.
The secretory IgA constitutes the main immunoglobulin in secretions
(ocular, salivary, mammary, tracheobronchial and urogenital
secretions), where it exists in the form of an IgA dimer combined
with another protein, the secretory component, which is probably
coiled around the IgA dimer and attached by disulfide bridges to
the CH2 domain of each IgA monomer. Unlike the J chain, the
secretory piece is not synthesized by the plasmocytes but by the
epithelial cells. The dimeric IgA secreted by the subepithelial
plasmocytes binds to the poly-Ig receptors present at the basal
pole of the epithelial cells. The s-IgA/receptor complex is then
endocytosed and transported through the cell while remaining
attached to the membrane of the transport vesicles. The latter fuse
with the plasma membrane at the luminal surface and release the
dimeric IgA combined with the secretory piece which results from
the cleavage of the receptor. Thus, the secretory piece facilitates
the transport of the IgAs in the secretions and protects them from
proteolysis.
Because of their capacity to cross the epithelium of the mucous
membranes and to prevent the entry of pathogens such as viruses,
bacteria, parasites and toxins, the IgAs play a major role in local
immunity: ocular, respiratory, digestive and urogenital immunity.
The mode of action of the IgAs encompasses active mechanisms
(complement activation, binding to the Fc receptor) and passive
mechanisms (blocking of the receptors for pathogens (viruses) and
inhibition of the motility of bacteria). A close correlation
between a specific IgA response and protection against an infection
has been demonstrated, in particular for viruses (rotavirus,
influenza virus, poliovirus, cytomegalovirus, respiratory syncytial
virus, Epstein-Barr virus). Class IgA protective antibodies
directed against numerous human pathogens (HIV, influenza A virus,
bacteria, toxins, parasites) have been isolated.
Because of this special property, IgAs have specific applications
for the diagnosis and treatment of infectious diseases and cancer.
They could be used in passive immunotherapy to neutralize pathogens
(serotherapy). They could also be used in active immunotherapy
(vaccination) as vector to target tumor antigens or antigens of
pathogenic microorganisms in the mucous membranes, so as to induce
local immunity specific to these antigens. In addition, they are
useful as reliable, safe, stable and well-defined reagent for the
diagnosis of diseases such as celiac disease, as a replacement for
human IgAs (antitransglutaminase, antiendomysium or antigliadin
IgA) obtained from patients, which expose technicians to risks of
transmission of human pathogens (virus, prion).
However, the development of these applications is limited because
there is no effective method for producing recombinant human or
humanized class IgA antibodies.
The expression humanized antibody is understood to mean an antibody
derived from a non-human mammal by fusion of the constant domains
of the heavy and light chains of a human antibody with the variable
domains of the heavy and light chains of an antibody from a
non-human mammal.
Indeed, the methods for producing recombinant human or humanized
antibodies which are currently available have the following
disadvantages: the in vitro methods are based on the simultaneous
expression, from one or more recombinant vectors, of antibody heavy
and light chains, of a J chain and optionally of a secretory piece;
the heavy and light chains comprise the variable domains of the
heavy and light chains (VH and VL) of a human or murine monoclonal
antibody of interest, fused respectively with the constant domains
CH1, CH2 and CH3 of a heavy chain .alpha., and C.lamda. or C.kappa.
of a human light chain, or the VH and VL domains are fused with a
CH3 domain including the C-terminal octapeptide (International
Applications PCT WO 98/30577 and PCT WO 99/54484). For example
International Application PCT WO 98/30577 describes the in vitro
production, with the aid of one or more recombinant baculoviruses,
of recombinant human dimeric mini-IgAs (IgA-J) comprising the VH
and VL domains of a murine or human monoclonal antibody, each fused
with a CH3 domain including the C-terminal octapeptide, combined by
means of a J chain; only one recombinant mini-IgA directed against
the HIV gp120, obtained from a class IgG1 neutralizing human
monoclonal antibody (S1-1 antibody), is described.
These methods, which are specific to IgAs, are limited to murine
antibodies and to a few rare human antibodies for which hybridomas
have been isolated. the in vivo methods are based on the production
of human monoclonal immunoglobulins from genetically modified mice
possessing a transgene consisting of: the complete IgH locus and
the locus of the kappa light chain, in their germinal
configuration, (PCT Application WO 02/059154, Mendez et al., Nature
Genetics, 1997, 15, 146-156; Green and Jakobovits, J. Exp. Med.,
1998, 188, 483-495 and American Patent U.S. application Ser. No.
08/759,620), a mini-IgH locus comprising one or more VH, DH and JH
genes, the C.mu. gene and a second gene for the constant region,
preferably for the C.gamma. region, and the locus of the kappa
light chain (PCT Application WO 02/059154, U.S. Pat. No.
5,545,807), and the complete IgH locus and the locus of the lambda
chain in its germinal configuration (American Patent U.S.
application Ser. No. 09/734,613). Said mice are optionally
genetically disabled for the endogenous kappa locus (.kappa.-/-
mice) and optionally possess a mutation which inactivates the
endogenous IgH locus (.mu.MT -/- mutation).
These methods do not make it possible to produce large quantities
of human class IgA immunoglobulins.
Surprisingly, the inventors have constructed transgenic mouse lines
which produce large quantities of humanized class IgA
immunoglobulins (in the gram per liter range in mice). The
antibodies produced by these animals are predominantly humanized
IgAs; they do not contain IgM and only very small quantities of
other classes of immunoglobulins (IgG and IgE).
Consequently, the subject of the invention is a non-human
transgenic mammal, characterized in that it comprises an IgH locus
modified by replacing the switch sequence S.mu. with all or part of
a transgene consisting of the C.alpha. gene for a human class A
immunoglobulin, including at least the exon encoding the CH3 domain
and the membrane exon.
In accordance with the invention, the C.alpha. transgene or the
part of this transgene including at least the exon encoding the CH3
domain and the membrane exon, which is inserted in place of the
switch sequence S.mu., is therefore located between the intronic
activator E.mu., in 5' and the C.mu. gene in 3' (FIG. 1).
In this construct, the suppression of the switch sequence S.mu.
associated with the insertion of the C.alpha. transgene in place of
this sequence, abolishes the expression of the endogenous .mu. gene
responsible for the synthesis of heavy IgM chains. In addition,
that of the other genes for the immunoglobulin heavy chains is
greatly reduced because of the blocking of the class switch toward
the immunoglobulin constant genes located downstream of C.mu. on
the endogenous IgH locus. Thus, the transgenic animals obtained
produce large quantities of chimeric IgAs in which the constant
domain of the heavy chain is humanized and the variable domains are
of murine origin.
The human transgenic .alpha. heavy chain benefits from a completely
diversified repertoire since it corresponds to the normal
repertoire generated by the rearrangements of the VH, D and JH
segments of the murine IgH locus. In addition, the transgenic
animals are capable of producing antibodies with high affinity as a
secondary response to the antigen since their B lymphocytes can
recruit the somatic hypermutation phenomenon.
According to an advantageous embodiment of the invention, said
non-human transgenic mammal is homozygous for said modified IgH
locus.
According to another advantageous embodiment of the invention, said
IgH locus is modified by replacing the switch sequence S.mu. with
the entire C.alpha. gene, including the CH1, CH2, CH3 and mb exons,
separated by the corresponding introns.
According to another advantageous embodiment of the invention, said
IgH locus is modified by replacing the switch sequence S.mu. with
the segment of the C.alpha. gene including the exon encoding the
CH3 domain and the membrane exon.
According to another advantageous embodiment of the invention, said
C.alpha. gene is C.alpha.1.
According to yet another advantageous embodiment of the invention,
said non-human transgenic mammal comprises another transgene
encoding a human immunoglobulin light chain.
According to an advantageous feature of this embodiment, said light
chain is a kappa chain.
Preferably, said transgene is a human kappa gene comprising the
intronic activator E.mu. upstream and the palindrome
hs3a/hs1,2/hs3b downstream. These sequences, which are described in
Chauveau et al., Gene, 1998, 222, 279-285, make it possible to
obtain a high expression of the human kappa chain in B cells and to
induce the somatic hypermutation of the human kappa transgene.
Preferably, said transgene is under the control of the promoter of
the human heavy chain (pVH).
According to another advantageous feature of this embodiment, said
non-human transgenic mammal is dizygous for said transgene.
According to an advantageous feature of the preceding embodiments
of the invention, said non-human transgenic mammals comprising
another transgene encoding a human kappa light chain possess an
endogenous locus of the immunoglobulin kappa light chain
inactivated (deleted or mutated) in particular by homologous
recombination. Preferably, said non-human transgenic mammals are
homozygous for said inactivation; preferably, they are transgenic
mice. Among the non-human transgenic mammals in which the
endogenous locus of the immunoglobulin kappa light chain has been
inactivated by homologous recombination, there may be mentioned in
particular the mouse line described in Zou et al., EMBO J., 1993,
12, 811-820.
Such non-human transgenic mammals produce humanized IgAs in which
practically all the light chains are of human origin.
According to another advantageous feature of the preceding
embodiments of the invention, said non-human mammals transgenic for
the .alpha.1 heavy chain and optionally for the human kappa light
chain possess an endogenous locus of the J chain inactivated
(deleted or mutated) in particular by homologous recombination.
Preferably, said non-human transgenic mammals are homozygous for
said inactivation; preferably, they comprise another transgene
encoding a human J chain; more preferably still, they are
transgenic mice. Such non-human transgenic mammals are humanized
both for the production of IgA and for a protein which combines
with the IgAs, the J chain.
The invention encompasses transgenic animals obtained from any
mammalian species.
According to another advantageous embodiment of the invention, said
non-human transgenic mammal is a transgenic mouse.
The invention encompasses in particular a double-transgenic mouse
line, called HAMIGA line for "Humanized Antibodies Made Up Of
Monoclonal Immunoglobulin A", comprising: an IgH locus modified by
replacing the switch sequence S.mu. with the C.alpha.1 gene for a
human class A immunoglobulin, and a complete V.kappa. gene
comprising the rearranged V.kappa.I gene with a J.kappa.5 gene, the
J.kappa.-C.kappa. intron and the C.kappa. gene, under the
transcriptional control of the promoter of the human heavy chain
(pVH), the intronic activator E.mu. upstream and the palindrome
hs3a/hs1,2/hs3b downstream.
The animals of this double-transgenic line produce IgAs that are
partially humanized for the heavy chain and completely humanized as
regards the light chain.
Indeed, the expression of the transgenic kappa chain in this line
is capable of causing allelic exclusion, that is to say of
preventing, in most transgenic B cells, the expression of the
endogenous genes for murine immunoglobulin light chains.
The repertoire of response to the antigens of this mouse line is
normal given that it is mainly the VH domain of the heavy chain
which contributes to the formation of the antibody site. Now, the
human transgenic .alpha. heavy chain benefits from a completely
diversified repertoire since it corresponds to the normal
repertoire generated by the rearrangements of the VH, D and JH
segments of the murine IgH locus, as specified above.
In addition, the mice of this transgenic line are capable of
producing antibodies with high affinity as a secondary response to
the antigen since their B lymphocytes can recruit the somatic
hypermutation phenomenon both at the level of the gene for the
heavy chain and the transgene for the kappa light chain.
The transgenic animals according to the invention are obtained by
conventional methods for animal transgenesis, according to the
standard protocols as described in Transgenic Mouse: Methods and
Protocols; Methods in Molecular Biology, Clifton, N.J., Volume 209,
October 2002, edited by: Marten H. Hofker, Jan Van Deursen, Marten
H. Hofker and Jan Van Deursen, published by Holly T. Sklar: Humana
Press.
The sequences of the human and murine genes for immunoglobulins
which serve for the construction of the transgenic animals
according to the invention are known and accessible in databases.
For example, the sequence of CH1, CH2 and CH3 exons and of the
membrane exon of the human C.alpha.1 gene correspond to the
accession numbers J00220 and M60326, respectively, in the
Genbank/EMBL database.
The construction of the V.kappa. gene is as described in Chauveau
et al., Gene, 1998, 222, 279-285; the sequence of the rearranged
V.kappa.I gene with the J.kappa.5 gene and the C.kappa. gene
corresponds to the sequence having the accession number X64133 in
the EMBL/Genbank database, which encodes a human light chain having
the sequence corresponding to the accession number CAA45494 in the
EMBL database.
The insertions of gene fragments into the genome of non-human
mammals may be carried out in a random manner, preferably they are
carried out in a targeted manner, by homologous recombination with
an appropriate targeting vector optionally comprising recombination
sequences of a site-specific recombinase such as the LoxP sites of
the Cre recombinase. The inactivations or deletions of gene
fragments in the genome of non-human mammals are carried out by
homologous recombination with an appropriate targeting vector
optionally comprising recombination sequences of a site-specific
recombinase such as the LoxP sites of the recombinase. The
double-transgenic animals are obtained by crossing animals
transgenic for the alpha heavy chain with animals transgenic for
the light chain, as defined above. The double-transgenic animals
are optionally crossed with transgenic animals in which the
endogenous locus of the immunoglobulin kappa light chain has been
inactivated by homologous recombination and/or with animals in
which the endogenous locus of the immunoglobulin J chain has been
inactivated and which additionally possess a human J transgene, as
defined above.
The subject of the present invention is also a homologous
recombination targeting vector, characterized in that it comprises
the C.alpha. gene for a human class A immunoglobulin or a segment
of this gene including at least the exon encoding the CH3 domain
and the membrane exon, flanked by fragments of sequences of the IgH
locus from a non-human mammal which are adjacent to the S.mu.
sequence.
According to an advantageous embodiment of said targeting vector,
it comprises a cassette for expressing an appropriate selection
marker, adjacent to said C.alpha. gene or to the segment of said
gene as defined above.
According to an advantageous feature of this embodiment, said
expression cassette is flanked by site-specific recombination
sequences. Preferably, said sequences are LoxP sequences of the Cre
recombinase. This feature optionally makes it possible to excise
said expression cassette.
According to another embodiment of said targeting vector, said
fragments of sequences which are adjacent to the S.mu. sequence are
of murine origin.
According to another embodiment of said targeting vector, the
C.alpha. gene or the segment of said gene is flanked in 5' by a
fragment of about 5 kb corresponding to the JH/E.mu. region and in
3' by a fragment of about 5 kb corresponding to the C.mu. region,
said fragments consisting of the sequences SEQ ID NO: 7 and 8
corresponding respectively to positions 131281 to 136441 and 140101
to 145032 in the sequence of murine chromosome 12 (accession number
AC073553 in the EMBL/GenBank database).
The subject of the present invention is also embryonic cells of a
non-human mammal, modified by a targeting vector as defined
above.
Said modified embryonic cells (totipotent stem cells) are useful
for the production of transgenic mammals as defined above; they are
injected into mammalian blastocysts, according to conventional
animal transgenesis techniques.
The subject of the present invention is also the use of a non-human
transgenic mammal as defined above for the production of humanized
class IgA antibodies or fragments of these antibodies.
The subject of the present invention is also a method for preparing
humanized class IgA antibodies or fragments of these antibodies,
characterized in that it comprises at least the following steps:
the immunization of a non-human transgenic mammal as defined above
with an antigen of interest, the production, by any appropriate
means, of humanized class IgA antibodies or fragments of these
antibodies, from serum, secretions or B lymphocytes of said
non-human transgenic mammal sacrificed beforehand.
The non-human transgenic mammals according to the invention have
the advantage of allowing the production of class IgA monoclonal
antibodies which are immediately humanized class IgA chimeric
antibodies. The method of producing humanized class IgA monoclonal
antibodies according to the invention is therefore more simple,
more rapid and more economical than the prior art methods since it
does not require additional steps of cloning the genes for said
antibodies and of fusing the variable domains of said antibodies
with the constant domains of human immunoglobulins.
The invention encompasses the production of polyclonal or
monoclonal antibodies consisting of monomeric or dimeric IgAs and
of s-IgAs, and fragments thereof, in particular the Fab, Fab'2 and
Fc fragments.
The humanized class IgA antibodies as defined above and fragments
thereof are prepared by conventional techniques known to persons
skilled in the art, such as those described in Antibodies: A
Laboratory Manual, E. Howell and D. Lane, Cold Spring Harbor
Laboratory, 1988.
More precisely: the polyclonal antibodies are prepared by
immunizing a non-human transgenic mammal as defined above with an
antigen of interest, optionally coupled to KLH or to albumin and/or
combined with an appropriate adjuvant such as Freund's (complete or
incomplete) adjuvant or aluminum hydroxide; after obtaining a
satisfactory antibody titer, the antibodies are harvested by
collecting serum from immunized animals and enriched with IgA by
precipitation, according to conventional techniques, and then the
specific IgAs are optionally purified by affinity chromatography on
an appropriate column to which the antigen is attached as defined
above, so as to obtain a preparation of monospecific IgAs. the
monoclonal antibodies are produced from hybridomas obtained by the
fusion of B lymphocytes from a non-human transgenic mammal as
defined above with myelomas, according to the Kohler and Milstein
technique (Nature, 1975, 256, 495-497); the hybridomas are cultured
in vitro, in particular in fermenters or produced in vivo, in the
form of ascites; alternatively, said monoclonal antibodies are
produced by genetic engineering as described in American U.S. Pat.
No. 4,816,567. For example, non-human transgenic mammals as defined
above are immunized strongly and repeatedly with chosen antigens
(bacterial, viral or fungal antigens, tumor-specific antigens such
as the carcinoembryonic antigen, and the like), according to a
standard protocol comprising a first immunization by
intraperitoneal injection of the antigen in an equivalent volume of
Freund's complete adjuvant and then a second immunization (booster)
15 days later under identical conditions but, this time, with
Freund's incomplete adjuvant. The monoclonal antibodies are
produced according to a standard protocol comprising sacrificing
the animals two weeks after the last booster, removing the spleen,
suspending the splenic lymphocytes and fusing these lymphocytes
with the SP2/0 cell line (this murine line does not produce any
murine antibody, is immortalized, and possesses the entire
secretion machinery necessary for the secretion of
immunoglobulins). the antibody fragments are produced from cloned
V.sub.H and V.sub.L regions, from mRNAs for hybridomas and for
splenic lymphocytes of an immunized non-human transgenic mammal
according to the invention; for example, the Fv and Fab fragments
are expressed at the surface of filamentous phages according to the
Winter and Milstein technique (Nature, 1991, 349, 293-299); after
several selection steps, the antibody fragments specific for the
antigen are isolated and expressed in an appropriate expression
system, by conventional techniques for cloning and expression of
recombinant DNA.
The antibodies or fragments thereof as defined above are purified
by conventional techniques known to persons skilled in the art,
such as affinity chromatography.
The subject of the present invention is also a humanized class IgA
antibody capable of being obtained by the method as defined above,
characterized in that it comprises a chimeric heavy chain in which
the constant domain(s) are of human origin and a human light chain
in which the variable domain is encoded by V.kappa.I-J.kappa.5.
The invention encompasses the humanized class IgA antibodies in
which the light chain is encoded by the V.kappa.I-J.kappa.5 gene
having the EMBL/Genbank sequence X64133 or a sequence produced by
hypermutation of this sequence, in particular after activation of B
lymphocytes in the presence of the antigen.
The subject of the present invention is also a fragment of a
humanized class IgA antibody capable of being obtained by the
method as defined above, characterized in that it comprises a
fragment of said heavy and light chains as defined above.
The invention encompasses polyclonal antibodies, monoclonal
antibodies and fragments thereof (Fab, Fc, Fab'2).
The humanized antibodies according to the invention and fragments
thereof as defined above are well tolerated in humans (minimization
of the risk of allergic reaction by interspecies immunization) and
have a prolonged half-life in humans, given that the constant
region of the heavy chain and the entire light chain of these
antibodies are of human origin.
The subject of the present invention is also a medicament
comprising a humanized class IgA antibody or a fragment of this
antibody, as defined above; such an antibody or its fragment is
used in particular in passive immunotherapy (serotherapy) for the
prevention and treatment of an infectious disease or cancer.
The subject of the present invention is also an immunogenic or
vaccine composition, characterized in that it comprises at least
one humanized class IgA antibody and a fragment of this antibody,
as defined above, combined with an antigen, preferably in the form
of an antigen-antibody complex comprising a humanized class IgA
antibody or a fragment of this antibody directed against said
antigen; such a composition makes it possible both to target the
antigen to the epithelium of the mucous membranes and to protect it
from proteolysis.
The subject of the present invention is also a pharmaceutical
composition, characterized in that it comprises at least one
humanized class IgA antibody or a fragment of this antibody, as
defined above, combined by any appropriate means with an active
ingredient; such a composition makes it possible both to target the
active ingredient to the epithelium of the mucous membranes and to
protect it from proteolysis.
According to an advantageous embodiment of the compositions
according to the invention, they additionally contain at least one
pharmaceutically acceptable vehicle and optionally carrier
substances and/or adjuvants.
The pharmaceutically acceptable vehicles, the carrier substances
and the adjuvants are those conventionally used.
The adjuvants are advantageously chosen from the group consisting
of oily emulsions, saponin, inorganic substances, bacterial
extracts, aluminum hydroxide and squalene.
The carrier substances are advantageously selected from the group
consisting of unilamellar liposomes, multilamellar liposomes,
miscelles of saponin or solid microspheres of a saccharide or
auriferous nature.
The compositions according to the invention are administered by the
general route (oral, intramuscular, subcutaneous, intraperitoneal
or intravenous) or by the local route (ocular, nasal, vaginal,
rectal); the dose and the rate of administration vary according to
the species (human or animal) and the disease to be treated.
The subject of the present invention is also a diagnostic reagent
comprising a humanized class IgA antibody or a fragment of this
antibody, as defined above.
The subject of the present invention is also the use of a humanized
class IgA antibody or a fragment of this antibody, as defined
above, for the preparation of a medicament intended for the
prevention and treatment of infectious diseases and cancer.
The subject of the present invention is also the use of a humanized
class IgA antibody or a fragment of this antibody, as defined
above, for the preparation of a reagent intended for the diagnosis
of infectious diseases and cancer.
In addition to the preceding features, the invention also comprises
other features which will emerge from the description which
follows, which refers to examples of production and use of
non-human transgenic mammals according to the present invention and
to the appended drawings in which:
FIG. 1 illustrates the structure of the modified IgH locus obtained
by homologous recombination between the murine IgH locus and the
targeting vector called p-alpha1KI, comprising a 5.5 kb fragment of
the human alpha 1 gene including three exons encoding the constant
domains CH1, CH2 and CH3 and the membrane (mb) exon and a neo
cassette bordered by LoxP sites (1.6 kb fragment), flanked upstream
by a fragment of about 5 kb corresponding to the JH-E.mu. region(DQ
52/JH fragment) and downstream by another fragment of about 5 kb
corresponding to the C.mu. gene (C.mu. fragment).
FIG. 2 illustrates the detailed structure of the targeting vector
called p-alpha1KI, comprising: a 5.5 kb fragment of the human alpha
1 gene including three exons encoding the constant domains CH1, CH2
and CH3 and the membrane (mb) exon and a neo cassette bordered by
LoxP sites (1.6 kb fragment), flanked upstream by a fragment of
about 5 kb corresponding to the JH-E.mu. region (DQ 52/JH fragment)
and downstream by another fragment of about 5 kb corresponding to
the C.mu. gene (C.mu. fragment).
FIG. 3 illustrates the confirmation of the sequence of the
targeting vector p-alpha1KI by enzymatic restriction with XhoI.
kH3: molecular weight marker. Lanes 3 and 4: clones comprising the
neo cassette inserted in the correct orientation; 5 fragments, 2 of
which co-migrate (5 kb and 5.3 kb), are detected: 6.4 kb (CH2+CH3
fragment of .alpha.1-neo cassette), 5 kb (C.mu. fragment), 5.3 kb
(JH fragment+CH1 fragment of al) and 3.7 kb (plasmid fragment+5'
DQ52 fragment). Lane 5: clone comprising the neo cassette inserted
in the reverse orientation; 4 fragments are detected: 9.5 kb (JH
fragment-CH2+CH3 fragment of .alpha.1-neo cassette), 5 kb (C.mu.
fragment), 3.7 kb (plasmid fragment+5' DQ52 fragment) and 2.4 kb
(CH1 fragment of .alpha.1+neo cassette).
FIG. 4 illustrates the Southern-blot profile of a recombinant
allele, compared with a wild-type allele; the genomic DNA digested
with EcoRI is hybridized with a probe located in 5' of the .delta.
gene.
FIG. 5 illustrates the Southern-blot analysis of the genomic DNA of
the ES clones transfected with the targeting vector p-alpha1KI; the
genomic DNA digested with EcoRI is hybridized with a probe
corresponding to the 5' region of the .delta. gene. The arrow
indicates a clone which has integrated the human al transgene by
homologous recombination (7.5 kb fragment corresponding to the
recombinant allele and 12 kb fragment corresponding to the
wild-type allele).
FIG. 6 illustrates the flow cytometry analysis of the expression of
a membrane receptor for the human IgA class at the surface of the
peripheral lymphocytes of homozygous animals of the transgenic line
alpha1KI. The x-axis represents the labeling with an anti-human
.alpha.1 antibody labeled with fluorescein and the y-axis
represents the labeling with an anti-murine CD19 antibody labeled
with phycoerythrin. The dotted rectangle indicates the cells
expressing both CD19 (B cells) and a human .alpha.1 heavy
chain.
FIG. 7 illustrates the flow cytometry analysis of the expression of
the human kappa light chain at the surface of the peripheral B
lymphocytes of mice of the kappa RNA line, compared with
nontransgenic mice (control). The x-axis represents the labeling
with the x-axis represents the labeling with an anti-human kappa
antibody labeled with fluorescein and the y-axis represents the
labeling with an anti-murine kappa antibody labeled with
phycoerythrin.
FIG. 8 illustrates the somatic hypermutation of the human kappa
transgene in the transgenic mouse line .kappa.RNA; the distribution
of the mutations along the human kappa light chain of 40 clones
isolated from B cells activated with PNA was analyzed. The
mutations generating an amino acid substitution, the silent
mutations and the mutations generating a stop codon are indicated
by .box-solid., .quadrature., and respectively. The amino acids
corresponding to the sites of hypermutation are indicated by their
nature and their position, and by the position of the mutation in
the codon (as a Roman numeral, in parentheses).
FIG. 9 illustrates the ELISA analysis of the specific human
chimeric IgA1 antibody response in the double-transgenic mice of
the HAMIGA line immunized with the ovalbumin antigen. The results
are expressed as arbitrary units of anti-ovalbumin IgA.
EXAMPLE 1
Production and Characterization of the Transgenic Line Alpha1KI
(Alpha1 Knock-In) Expressing a Chimeric Human Immunoglobulin Alpha
1 Heavy Chain
The human alpha 1 gene, including the three exons encoding the
constant domains CH1, CH2 and CH3 and the membrane (mb) exon, was
inserted by homologous recombination, in place of the switch region
S.mu. of the murine heavy chain (S.mu.), so as to block the class
switch to the constant genes for immunoglobulins located downstream
of C.mu. on the endogenous locus (murine IgH locus, FIG. 1). The
targeted region abolishes the expression of the endogenous .mu.
gene responsible for the synthesis of IgM heavy chains, and greatly
reduces that of other genes for immunoglobulin heavy chains.
Consequently, the transgenic line obtained produces a large
quantity of chimeric IgAs in which the humanized constant domain
corresponds to the IgA1 isotype.
1) Construction of the Homologous Recombination Targeting
Vector
The plasmid constructs were produced from the plasmid bluescript SK
(pSK) (STRATAGENE) and from the bacterial strain E.coli
TG1(STRATAGENE), using the conventional protocols for the
preparation, cloning and analysis of DNA such as those described in
Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000,
Wiley and Son Inc, Library of Congress, USA).
The homologous recombination vector or targeting vector derived
from pSK, called p-alpha1KI (FIG. 2), comprises: a 5.5 kb fragment
of the human alpha 1 gene including three exons encoding the
constant domains CH1, CH2 and CH3 and the membrane (mb) exon and a
neo cassette (1.6 kb fragment), flanked upstream by a fragment of
about 5 kb corresponding to the JH-E.mu. region (DQ 52/JH fragment)
and downstream by another fragment of about 5 kb corresponding to
the C.mu. gene (C.mu. fragment).
More specifically, the various fragments were inserted into the
plasmid bluescript SK, according to the following steps: In a first
step, the C.mu. fragment corresponding to positions 140101 to
145032 of murine chromosome 12 (Genbank/EMBL AC073553) was
amplified by PCR with the aid of appropriate specific primers and
then cloned at the XhoI site of pSK to give the plasmid pA. In a
second step, the DQ 52/JH fragment corresponding to positions
131281 to 136441 of murine chromosome 12 (Genbank/EMBL AC073553)
was amplified by PCR with the aid of appropriate specific primers
and then cloned in 5' of the C.mu. fragment, between the EcoRV and
ClaI sites of the plasmid pA, to give the plasmid pB. In a third
step, the neo cassette described in Pinaud et al., Immunity, 2001,
15, 187-199 was inserted at the SalI site between DQ52/JH and
C.mu., to give the plasmid pC.
The SacI-BamHI fragment of 5.5 kb of a recombinant plasmid
comprising the entire human alpha 1 gene, including the exon
sequences CH1, CH2 and CH3 (Genbank/EMBL J00220) and the membrane
exon (Genbank/EMBL X64133) was ligated at each of its ends with
ClaI adaptors.
Finally, in a final step, the 5.5 kb fragment flanked with ClaI
adaptors thus obtained was inserted between the JH fragment and the
neo cassette at the ClaI site of the plasmid pC to give the
targeting vector called p-alpha1KI.
The p-alpha1KI sequence was verified by automated sequencing and by
restriction analysis with the enzymes ClaI and XhoI (FIG. 3).
2) Transfection of ES Cells and Injection Into Blastocysts
The clones of ES cells derived from the 129/SJ line were isolated,
analyzed and then injected into blastocysts of C57/Black 6 mice
using conventional protocols for transgenesis and analysis of
genomic DNA, such as those described in Current Protocols in
Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and Son Inc,
Library of Congress, USA).
More specifically, ES cells were transfected by electroporation of
the p-alpha1KI DNA linearized at the NotI site. The clones selected
in the presence of geneticin were collected and the genomic DNA
digested with EcoRI was analyzed by Southern blotting with the aid
of a radioactive probe hybridizing outside the site of homologous
recombination, in 5' of the constant Delta (.delta.) gene and of
its EcoRI site (FIG. 4); this probe, amplified by PCR with the aid
of appropriate specific primers, corresponds to positions 140101 to
145032 of the murine chromosome 12 sequence (EMBL/Genbank
AC073553).
The presence of a recombinant allele is visualized with a fragment
of about 7.5 kb (representing the murine .mu. fragment and the neo
cassette) whereas the wild-type allele corresponds to a fragment of
12 kb (FIG. 5). Under these conditions, out of 303 clones analyzed,
4 proved positive.
Verification of the karyotype of two of the four recombinant clones
showed no chromosomal abnormality (aneuploidy).
These clones were injected into blastocysts of C57/Black 6 mice
using conventional transgenesis protocols such as those described
in Transgenic Mouse: Methods and Protocols, cited above. Among the
mice obtained, those exhibiting the highest degree of chimerism
were analyzed by PCR and by ELISA. A mouse line homozygous for the
recombinant IgH locus, called hereinafter alpha 1 knock-in or
alpha1KI line, was then obtained by crossing heterozygous animals
exhibiting the highest degree of chimerism.
3) Detection of the Recombinant IgH Locus Carrying the Human
C.alpha.1 Gene (Alpha 1 Knock-in or Alpha1KI Allele) and of the
Wild-type IgH Locus (Wild-type .mu. Allele) By PCR
The genomic DNA of a tail sample from homozygous animals obtained
as specified above was analyzed by PCR with the aid of the
following two pairs of primers: pair specific for the non-mutated
murine IgH locus (wild-type .mu. allele):
TABLE-US-00001 UpstreamSpe I Smu primer: (SEQ ID NO: 1) 5' GAG TAC
CGT TGT CTG GGT CAC 3' SacI-3' Imu primer: (SEQ ID NO: 2) 5' GAG
CTC TAT GAT TAT TGG TTA AC 3'
The amplification reaction was carried out with a hybridization
temperature of 61.degree. C. This PCR amplifies in 30 cycles a
fragment of 91 base pairs delimiting the SpeI site specific for the
nonmutated murine IgH locus. pair specific for the recombinant IgH
locus carrying the human C.alpha.1 gene (alpha 1 knock-in or
alpha1KI allele):
TABLE-US-00002 NeoI primer: (SEQ ID NO: 3) 5' GCA TGA TCT GGA CGA
AGA GCA T 3' Neo2 primer: (SEQ ID NO: 4) 5' TCC CCT CAG AAG AAC TCG
TCA A 3'
The amplification reaction was carried out with a hybridization
temperature of 55.degree. C. This PCR amplifies in 30 cycles a
fragment of 120 base pairs specific for the recombinant IgH locus
carrying the human C.alpha.1 gene (alpha 1 knock-in or alpha1KI
mutation).
A mouse line homozygous for the alpha1KI mutation, called
hereinafter alpha 1 knock-in or alpha1KI line, was established; the
animals of this line are systematically and simultaneously negative
in PCR with the primers specific for the wild-type .mu. allele and
positive in PCR with the primers specific for the alpha 1 knock-in
allele.
4) Assay of Total Serum IgAs By Nephelometry and By ELISA
a) Nephelometry
The serum IgAs were assayed by nephelometry on an automated mchine
BNII.TM. (BEHRING) using the IgA assay kit (BEHRING), according to
the supplier's recommendations.
The assay of the serum IgAs gave results which correlated fully
with those of the genotyping carried out by PCR: the non-mutant
control animals have a zero level of human class IgA
immunoglobulins the heterozygous animals .alpha.1-KI also have an
undetectable level of human IgAs and a normal level of murine IgMs
the homozygous animals .alpha.1-KI have a significant level of
human IgAs, this level varying between 0.4 and 0.6 g/l in the
serum. On the other hand, the murine IgMs are undetectable in the
serum of these animals. b) ELISA
The results obtained by nephelometry were confirmed by ELISA
according to the following steps: 96-well plates (Maxisorb.TM.,
NUNC) were coated either with non-labeled anti-human IgA antibodies
or with non-labeled anti-murine IgM antibodies by incubating
overnight at +4.degree. C. in the presence of goat Fab'2 anti-human
IgAs or anti-murine IgMs (Southern Biotechnologies Associates),
diluted 1/500 in 0.1 M carbonate buffer, pH 8.3 (100
microliters/well). After 3 washings with PBS buffer containing 0.1%
Tween (PBS-Tween 0.1%), the plates were saturated in the presence
of PBS containing 10% fetal calf serum (100 microliters/well).
After 3 washings with PBS-Tween 0.1% buffer, the sera to be tested,
diluted 1/100 and 1/500 in PBS buffer containing 10% fetal calf
serum were added (100 microliters/well) and the plates were
incubated for 3 hours at 37.degree. C. After 3 washings with
PBS-Tween 0.1% buffer, an anti-human IgA antiserum labeled with
alkaline phosphatase or an anti-murine IgM serum labeled with
alkaline phosphatase (Biosys) diluted 1/1000 in PBS-Tween 0.1% (100
microliters/well) were added and the plates were incubated for 1
hour at 37.degree. C. After 3 washings with PBS-Tween 0.1% buffer,
the bound IgAs and IgMs were visualized by adding alkaline
phosphatase substrate (p-nitrophenyl phosphate, SIGMA) at 1 mg/ml
in 0.2M Tris buffer, pH 7.0. The reaction was blocked by adding
0.5N sodium hydroxide (50 microliters/well) and then the absorption
was measured at a wavelength of 405 nm.
The quantitative data are obtained by extrapolation with a series
for a standard serum (BEHRING) for the assay of the human IgAs, and
for a murine monoclonal IgM (SOUTHERN BIOTECHNOLOGIES ASSOCIATES)
for the assay of the murine IgMs.
The assay of the serum IgAs by ELISA shows a significant difference
between the homozygotes and the heterozygotes; the sera of
homozygotes contain 0.4 and 0.6 g/l of IgA1, whereas zero or very
low absorbance values are observed for the sera of heterozygotes
even at the lowest dilution (1/100). By contrast, when the murine
IgMs are assayed by ELISA, a normal murine IgM level is observed
(of the order of 1 g/l) in the "non-mutant" control mice and in the
animals heterozygous for the .alpha.1-KI mutation. On the other
hand, the murine IgM level is zero in the animals homozygous for
the .alpha.1-KI mutation.
5) Investigation of the Expression of a Membrane Receptor for the
Human IgA Class at the Surface of the Peripheral Lymphocytes of
Mutant Animals
The homozygous animals carrying the .alpha.1-KI mutation were
phenotyped by flow cytometry, by double labeling with the aid of
antibodies specific for human IgA1 or murine IgM labeled with
fluorescein, and of antibodies specific for B cells (anti-CD19
antibodies) labeled with phycoerythrin. More specifically:
Preparation of lymphoid cells: two peripheral lymphoid organs: the
spleen and the Peyer's patches, were removed separately from
homozygous mutant animals .alpha.1KI, dilacerated in a versene
buffer (Invitrogen), and filtered on sieve (40 microns) in order to
obtain a suspension of individual cells freed of cellular
aggregates. The spleen cells were then centrifuged and subjected to
an additional step of osmotic shock in order to lyse the red blood
cells by resuspending the cellular pellet in 1 ml of distilled
water. The cells of the samples were then immediately resuspended
in complete medium (RPMI+10% fetal calf serum), counted and stored
on ice. Labeling with the aid of fluorescent antibodies: 10.sup.5
cells from each sample were incubated for 30 minutes at 4.degree.
C. with a 1/100 dilution, either of an anti-mouse IgM antibody
labeled with fluorescein isothiocyanate (Southern Biotechnologies),
or of an anti-human IgA antibody labeled with fluorescein
isothiocyanate, or alternatively the combination of one of the
preceding antibodies with an antibody specific for B cells
(anti-CD19 antibodies) labeled with phycoerythrin (double
labeling). The cells were then washed in 5 ml of PBS and then the
supernatant was separated after decantation and the cells were
resuspended in 100 microliters of PBS, 0.5% BSA, 0.1 mM EDTA.
Cytofluorimetric analysis: the labeled cells were analyzed by flow
cytometry (COULTER XL.TM.).
The results of the flow cytometry are in agreement with those of
the assay of serum immunoglobulins. In the homozygous animals of
the alpha1-KI line, no expression of murine IgMs is detected either
in the spleen or in the Peyer's patches.
Yet in the absence of expression of IgM, a compartment of CD19+
peripheral B cells is capable of becoming differentiated in these
animals and represents 10 to 12% of the spleen lymphocytes or 40 to
60% of the lymphocytes of the Peyer's patches. This compartment
expresses membrane IgAs in which the humanized heavy chain is
recognized by an antibody specific for the IgAls and labeled with
fluoroscein (FIG. 6).
EXAMPLE 2
Production and Characterization of the Transgenic Line .kappa. RNA
Expressing a Human Immunoglobulin Kappa Light Chain
A transgenic animal line expressing in all their B cells a human
kappa light chain encoded by the variable region
V.kappa.I-J.kappa.5 and the C.kappa. region (kappa RNA chain,
EMBL/Genbank X64133) was obtained by direct transgenesis from the
expression vector described in Chauveau et al., Gene, 1998, 222,
279-285.
1) Construction of the Transgenesis Vector
The transgenesis vector is the plasmid pALIE.mu. described in
Chauveau et al., Gene, 1998, 222, 279-285; it contains both the VH
promoter and the E.mu. enhancer in 5' of the cassette encoding the
kappa RNA chain, and in 3' of this cassette: the three enhancers
hs3a, hs12 and hs3b, located in 3' of the IgH locus in 3'. The
coding sequence corresponds to the V.kappa.I-J.kappa.5-C.kappa.
chain (Genbank/EMBL X64133). The plasmid pALIE.mu. was linearized
with the restriction enzymes NotI and PvuI which cut inside the
plasmid sequence, NotI being located upstream of the promoter which
precedes the cloned V.kappa. segment and PvuI being located within
the ampicillin resistance gene carried by the plasmid. The fragment
including the entire kappa expression cassette flanked by all the
promoter and regulatory elements for expression was then randomly
inserted into mouse blastocysts using conventional direct
transgenesis protocols such as those described in Transgenic Mouse:
Methods and Protocols, cited above.
2) Identification of the Founder Animals of the .kappa. RNA Line
and Typing of Their Progeny
A transgenic mouse line possessing the .kappa. RNA transgene was
obtained after injection of the expression vector; the presence of
this human transgene was verified on the DNA of the mice by
Southern blotting with the aid of a probe specific for the human
C.kappa. region (EcoRI-EcoRI fragment of 2.5 kb including the
entire human C.kappa. exon). The animals carrying the insert of the
transgene on the two alleles of the site of insertion (homozygous
animals) have a double quantity of transgene and can be
distinguished by Southern blotting from the animals carrying a
single copy of the transgene (heterozygous animals). Alternatively,
the presence of the transgene was detected by PCR with the aid of
primers which make it possible to specifically amplify the human
sequence V.kappa.I-J.kappa.5-C.kappa. (Genbank/EMBL X64133).
3) Investigation of the Expression of the Human Kappa Light Chains
at the Surface of the Peripheral Lymphocytes of Mice of the Kappa
RNA Line
The dizygous animals carrying the kappa RNA transgene were
phenotyped by flow cytometry, by double labeling with the aid of an
anti-murine .kappa. antibody (labeled with phycoerythrin) in
conjunction with an anti-human .kappa. antibody (labeled with
fluorescein isothiocyanate), according to the protocol as described
in example 1.
These animals show an expression of the human .kappa. transgene on
the majority of the B cells (FIG. 7). Furthermore, the transgene
induces a phenomenon of allelic exclusion such that the B cells
expressing the human .kappa. transgene do not express an endogenous
gene for the mouse light chains. By cytometry, these cells are
therefore positive during labeling with the anti-human .kappa.
chain antiserum and negative with the anti-murine .kappa. chain
antiserum (FIG. 7).
4) Analysis of the Somatic Hypermutation of the .kappa. Transgene
in Mice of the Kappa RNA Line
It has been shown that this human .kappa. light chain is capable of
combining with heavy chains and of becoming diversified by virtue
of the phenomenon of somatic hypermutation (triggered by a response
to the antigen). This transgene which preserves the endogenous
architecture of a .kappa. gene with presence of the
J.kappa.-C.kappa. intron between V.kappa.J.kappa. and C.kappa.,
further benefits from a high expression provided by the P.sub.VH
promoter/E.mu. enhancer+regulatory palindrome 3'IgH (hs3a, hs1,2,
hs3b) combination. The cumulative action of all these regulatory
elements makes it possible to recruit the somatic hypermutation
machinery at the level of the transgene. More specifically, the
Peyer's patches of transgenic mice are removed by dissection of the
intestine. The cellular suspension is prepared by grinding the
Peyer's patches through a nylon membrane. The cells are washed
three times at +4.degree. C. in DMEM containing 10% fetal calf
serum. The dead cells were removed after each washing and the
cellular suspension was adjusted to 10.sup.6 cells/ml.
The cells were incubated for 30 min at +4.degree. C. in the
presence of biotinylated anti-B220 antibodies. After two washings
with DMEM containing 5% fetal calf serum, the cells were incubated
for 30 min at +4.degree. C. in the presence of streptavidin coupled
to phycoerythrin, and then washed and resuspended in PBS containing
5% fetal calf serum. After adding a lectin specific for the
activated B cells (PNA for peanut agglutinin) conjugated with FITC,
the cellular suspension was incubated for 30 min at +4.degree. C.
After two washings with DMEM, the cells were resuspended in DMEM
and then they were sorted, by flow cytometry, into two populations:
B220.sup.+PNA.sup.high (activated B) and B220.sup.+PNA.sup.low
(resting B).
The genomic DNA was extracted from the two cellular populations
sorted with the aid of the kit QIAamp Tissue (QIAGEN).
Amplification by polymerase chain reaction (PCR) was carried out on
2 .mu.l of genomic DNA using primers corresponding to the signal
region of the human V.kappa.1 (5'-AAGTCGACATGGACATGAGGGTGCC-3')
(SEQ ID NO:5) and at the beginning of the human J.kappa.5 region
(5'-TTCTCGAGACTTAGGTTTAATCTCCAG-3') (SEQ ID NO:6). The
amplification program consisted of: an initial step of denaturation
at 94.degree. C. for 5 min; followed by 35 cycles consisting of a
denaturation step at 94.degree. C. for 30 s, a hybridization step
at 52.degree. C. for 30 s and an extension step at 72.degree. C.
for 30 s; and then a final extension step at 72.degree. C. for 7
min.
The amplification product was purified on 1.2% agarose gel, eluted
(kitQIAquick Gel Extraction kit, QUIAGEN) and then cloned into the
vector pCRII-TOPO (INVITROGEN). The recombinant clones were tested
by enzyme restriction and then purified (Flexiprep kit, PHARMACIA)
and sequenced by the Sanger method. The sequencing reactions were
carried out by PCR with the aid of the primers M13 reverse and
M13(-20) and fluorescent dideoxynucleotides and then analyzed by
capillary electrophoresis on an automated sequencer (ABI-PRISM 310,
PERKIN-ELMER). The sequences obtained from the activated B cells
were then aligned with the original sequence of the non-mutated
transgene (Genbank/EMBL X64133). The number and the position of the
mutations were analyzed (FIG. 8).
The .kappa. transgene undergoes this somatic hypermutation at a
rate which is practically as high (17 mutations per 1000 bases) as
the endogenous immunoglobulin genes (which mutate at a rate of 40
mutations per 1000 bases). This single transgene is therefore
capable of generating a kappa "repertoire" having some
diversity.
EXAMPLE 3
Production and Characterization of the Double-transgenic HAMIGA
Line Expressing a Chimeric Alpha 1 Heavy Chain and a Kappa Light
Chain of Human Immunoglobulins
The crossing of the KRNA and alpha1-KI lines described in the
preceding examples generates double transgenic KRNA/alpha1-KI
mice.
To do this, the animals homozygous for the alpha1-KI mutation and
homozygous for the .kappa. RNA transgene were crossed with each
other. In the first generation (F1) after this crossing, all the
animals obtained are heterozygous for the alpha1-KI mutation and
heterozygous for the .kappa. RNA transgene. These F1 animals were
therefore crossed again: in the next generation (F2) the laws of
Mendelian genetics make it possible to obtain 1 animal out of 4
homozygous for the alpha1-KI mutation and one animal out of 4
homozygous for the .kappa. RNA transgene. Among these F2 animals,
one animal out of 16 could therefore be selected as carrying both
the alpha1-KI mutation in the homozygous state and carrying the
.kappa. RNA transgene in the homozygous state. These animals are
the founders of the HAMIGA line and they stably transmit to their
progeny the genes which simultaneously allow the production of a
humanized alphal heavy chain in place of the production of murine
IgMs and the production and diversification by hypermutation of a
human .kappa. chain.
This double transgenic mouse line is called HAMIGA line for
"Humanized Antibodies Made Up Of Monoclonal Immunoglobulin A".
1) Production of the Double-transgenic HAMIGA Line
a) Presence of the .kappa. Transgene
The transmission of the .kappa. RNA transgene during crossings of
transgenic animals was monitored by Southern blotting with the aid
of a probe specific for the human C.kappa. region (EcoRI-EcoRI
fragment of 2.5 kb including the entire human C.kappa. exon).
The expression of the .kappa. transgene in the mutant animals was
detected by ELISA assay of free human kappa chains eliminated in
the urine of the animal. More specifically: 96-well plates
(Maxisorb.RTM., NUNC) were incubated overnight at +4.degree. C. in
the presence of a non-labeled anti-human .kappa. antibody
(Kallestad) diluted 1/1000 in 0.1M carbonate buffer pH 8.3 (100
microliters/well). After 3 washings with PBS buffer containing 0.1%
Tween (PBS-Tween 0.1%), the plates were saturated in the presence
of PBS containing 10% fetal calf serum (100 microliters/well).
After 3 washings with PBS-Tween 0.1% buffer, the urine samples to
be tested, diluted 1/100 and 1/500 in PBS buffer containing 10%
fetal calf serum were added (100 microliters/well) and the plates
were incubated for 3 hours at 37.degree. C. After 3 washings with
PBS-Tween 0.1% buffer, an anti-human .kappa. antiserum labeled with
alkaline phosphatase (SIGMA) diluted 1/1000 in PBS-Tween 0.1% (100
microliters/well) was added and the plates were incubated for 1
hour and at 37.degree. C. After 3 washings with PBS-Tween 0.1%
buffer, the bound human kappa light chains were visualized by
adding alkaline phosphatase substrate (p-nitrophenyl phosphate,
SIGMA) at 1 mg/ml in 0.2 M Tris buffer, pH 7.0. The reaction was
blocked by adding 0.5N sodium hydroxide (50 microliters/well) and
then the absorption was measured at a wavelength of 405 nm.
Alternatively, the expression of the human kappa transgene was
analyzed by flow cytometry as described in example 2. The results
show that the presence of the .kappa. RNA transgene causes an
important phenomenon of allelic exclusion, such that among the
peripheral lymphocytes more than 50% express the human light chain
and do not therefore rearrange the genes for the murine light
chains in order to express a murine light chain.
b) Homozygosity for the .alpha.1-KI Mutation
The first single element indicating .alpha.1-KI homozygocity is the
presence of a high level of human IgA1s in the serum of the
animals. In addition, the homozygocity was confirmed by PCR by the
positivity of the ".alpha.1-KI PCR" combined with the negativity of
the "wild-type .mu. allele PCR". Finally, after sacrificing the
animals, flow cytometry analysis made it possible to show on the
lymphocytes of the spleen and of the Peyer's patches that the
entire B lymphocytes (CD19+) express membrane human IgA1s whereas
in parallel no B cell expresses murine IgM.
c) Verification of the Simultaneous Presence of the Alpha1-KI
Mutation and of the .kappa. RNA Transgene in the HAMIGA Animals and
Their Progeny
The double-transgenic HAMIGA animals were characterized as those
simultaneously corresponding to the two specificities described
above: the presence of the K RNA transgene in the homozygous state
and the homozygocity for the alpha1-KI mutation. Furthermore, these
animals reproduce while preserving these two specificities and the
phenotype of their progeny has the following properties,
simultaneously and in a stable manner: the production of humanized
IgA1 at a sizable level (easily verifiable by ELISA or nephelometry
on a simple blood sample taken from live animals at the level of
the retro-orbital sinus) the production of human .kappa. light
chain (easily verifiable by ELISA on a simple urine sample taken
from live animals). 2) Immunization of the Animals
The animals were immunized once by intraperitoneal injection of 10
micrograms of ovalbumin (SIGMA) diluted in 100 microliters of
physiological saline and emulsified with 200 microliters of
Freund's complete adjuvant (SIGMA).
After 4 weeks, the animals were subjected to a vaccine booster by
intraperitoneal injection of 10 micrograms of ovalbumin (SIGMA)
diluted in 100 microliters of physiological saline and emulsified
with 200 microliters of Freund's incomplete adjuvant (SIGMA).
3) Assay of the Antibodies Specific for the Vaccine Antigen
(Ovalbumin)
The presence of antibodies specific for the vaccine antigen
ovalbumin was analyzed by ELISA 4 weeks, and then 7 weeks after the
second injection of the antigen, according to the following
technique: 96 well-plates (Maxisorb.RTM., NUNC) were incubated
overnight at +4.degree. C. in the presence of ovalbumin at the
concentration of 10 micrograms/ml in 0.1M carbonate buffer pH 8.3
(100 microliters/well). After 3 washings with PBS buffer containing
0.1% Tween (PBS-Tween 0.1%) the plates were saturated in the
presence of PBS containing 10% fetal calf serum (100
microliters/well). After 3 washings with PBS-Tween 0.1% buffer, the
serum samples to be tested, diluted 1/20 and 1/100 in PBS buffer
containing 10% fetal calf serum were added (100 microliters/well)
and the plates were incubated for 3 hours at 37.degree. C. After 3
washings with PBS-Tween 0.1% buffer, an anti-human IgA antiserum
labelled with alkaline phosphatase (BIOSYS) diluted 1/1000 in
PBS/Tween 0.1% (100 microliters/well) was added and the plates were
incubated for 1 hour at 37.degree. C. After 3 washings with
PBS-Tween 0.1% buffer, the bound human kappa light chains were
visualized by adding alkaline phosphatase substrate (p-nitrophenyl
phosphate, SIGMA) at 1 mg/ml in 0.2M Tris buffer, pH 7.0. The
reaction was blocked by adding 0.5N sodium hydroxide (50
microliters/well) and then the absorption was measured at a
wavelength of 405 nm. The level of anti-ovalbumin IgA antibodies
was expressed as arbitrary units established for sera diluted 1/100
as a function of the Optical density tested serum/Optical density
control serum ratio.
The results presented in FIG. 9 show the presence of antibodies
specific for the vaccine antigen ovalbumin 4 weeks (level of human
anti-ovalbumin IgA1 antibodies at 388 units), and then 7 weeks
after the second injection of the antigen (level of anti-ovalbumin
IgA antibodies at 162 units). In parallel, it was also verified
that in the absence of immunization of the animals, the level of
the anti-ovalbumin IgA antibodies detected remained less than 30
units.
The repertoire of response to the antigens of these mice is
expected as subnormal since it is known that it is essentially the
VH domain of the heavy chain which contributes to the formation of
the antibody site (yet the human transgenic al heavy chain benefits
from a completely diversified repertoire since it corresponds to
the normal repertoire generated by the rearrangements of the VH, D
and JH segments of the murine IgH locus). These mice are capable of
producing antibodies of high affinity as a secondary response,
which results from the fact that their B lymphocytes can recruit
the phenomenon of somatic hypermutation both at the level of the
heavy chain gene and of the .kappa. RNA light chain transgene.
As is evident from the above, the invention is not at all limited
to its embodiments, implementations and applications which have
just been described more explicitly; it embraces on the contrary
all the variants which may occur to the specialist in this field,
without departing from the framework or the scope of the present
invention.
SEQUENCE LISTINGS
1
8121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1gagtaccgtt gtctgggtca c 21223DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2gagctctatg attattggtt aac 23322DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 3gcatgatctg gacgaagagc at
22422DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4tcccctcaga agaactcgtc aa 22525DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5aagtcgacat ggacatgagg gtgcc 25627DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 6ttctcgagac ttaggtttaa
tctccag 2775161DNAMus musculus 7acaggcctga gagaacagac tctggaaata
gatgggactt acggagctaa gatctagagc 60tcatctacag agcagaatcc cagccaagag
aacaaagaat actgactctc tcctgttccc 120tactcctaga gttctaaaac
acactatagg gaagggagcc tctagacctc cgtccattcc 180ccatcttgct
cattccatct tcccatgtcc ccaggtctcc aagccacaga caccaccttt
240cctattcacc cacctttctg tgtccctagg tccccaggcc atagtcacct
ccccccacac 300cccgctcacc ctgccccatc tatgccccta gatgcttact
taccagagtc ttttgtctga 360cgtggggcta caagcatcta tgctccctaa
gcacctactg ctgacctgta ggacccagct 420ctgaaccaac tcatataagt
aaatacagac tctcccctgt cttaggatgg ccccctgggt 480caggaggaga
ccactgccaa ggaaccttct cttagagcac tgaactcctc ccctgtacca
540cttaggacag acctgagacc tattattact gattaccaga gctctggcag
tgaccacgga 600ggagatagat ccaccctgga cacaggaaac acagcaccag
agatactgct tcatcacaac 660agtagagtga cactttagac tttaatttgg
gtcactttcc tgctgtagag gtgggatcag 720aaagcaaaga gcagtatgag
tgcctgatag gcacccaagt acactataga gtactcatgg 780tgaataaggt
acctccatgg cttcccaggg aggggcactg ccccaccccc accatcacag
840acctttctcc atagttgata actcagacac aagtgaatga cagatggacc
tccatctgct 900cttattttaa aaagaagaca aaccccacag gctcgagaac
tttagcgact gttttgagag 960aaatcattgg tccctgactc aagagatgac
tggcagattg gggatcagaa tacccatact 1020ctgtggctag tgtgaggttt
aagcctcaga gtccctgtgg tctctgactg gtgcaaggtt 1080ttgactaagc
ggagcaccac agtgctaact gggaccacgg tgacacgtgg ctcaacaaaa
1140accttctgtt tggagctctc caggggcagc ctgagctatg aggaagtaga
gaggcttgag 1200aaatctgagg aagaaaagag tagatctgag aggaaaggta
gctttctgga ggtcaggaga 1260cagtgcagag aagaacgagt tactgtggac
aggtcttaga tggggaaaga atgagcaaat 1320gcaagcatca gaagggtgga
tgcaatgtcc tgccaaggac ttaccaagag gatccccgga 1380cagagcaggc
aggtggagtt gactgagagg acaggatagg tgcaggtccc tctcttgttt
1440cctttctcct tctcctgttt ccttcttctc ttgtcacagg tctcactatg
ctagccaagg 1500ctagcctgaa agattaccat cctacagatg ggcccatcca
gttgaattaa ggtggagatc 1560tctccaaaca tctgagtttc tgaggcttgg
atgccactgg ggacgccaag ggactttggg 1620atgggtttgg ttggccccag
atgaagggct acttcactgg gtctataatt actctgatgt 1680ctaggaccag
ggggctcagg tcactcaggt caggtgagtc ctgcatctgg ggactgtggg
1740gttcaggtgg cctaaggcag gatgtggaga gagttttagt ataggaacag
aggcagaaca 1800gagactgtgc tactggtact tcgatgtctg gggcacaggg
accacggtca ccgtctcctc 1860aggtaagctg gcttttttct ttctgcacat
tccattctga aacgggaaaa gatattctca 1920gatctcccca tgtcaggcca
tctgccacac tctgcatgct gcagaagctt ttctgtaagg 1980atagggtctt
cactcccagg aaaagaggca gtcagaggct agctgcctgt ggaacagtga
2040caatcatgga aaataggcat ttacattgtt aggctacatg ggtagatggg
tttttgtaca 2100cccactaaag gggtctatga tagtgtgact actttgacta
ctggggccaa ggcaccactc 2160tcacagtctc ctcaggtgag tccttacaac
ctctctcttc tattcagctt aaatagattt 2220tactgcattt gttggggggg
aaatgtgtgt atctgaattt caggtcatga aggactaggg 2280acaccttggg
agtcagaaag ggtcattggg agccctggct gacgcagaca gacatcctca
2340gctcccatac ttcatggcca gagatttata gggatcctgg ccagcattgc
cgctaggtcc 2400ctctcttcta tgctttcttt gtccctcact ggcctccatc
tgagatcatc ctggagccct 2460agccaaggat catttattgt caggggtcta
atcattgttg tcacaatgtg cctggtttgc 2520ttactggggc caagggactc
tggtcactgt ctctgcaggt gagtcctaac ttctcccatt 2580ctaaatgcat
gttgggggga ttctgggcct tcaggaccaa gattctctgc aaacgggaat
2640caagattcaa cccctttgtc ccaaagttga gacatgggtc tgggtcaggg
actctctgcc 2700tgctggtctg tggtgacatt agaactgaag tatgatgaag
gatctgccag aactgaagct 2760tgaagtctga ggcagaatct tgtccagggt
ctatcggact cttgtgagaa ttaggggctg 2820acagttgatg gtgacaattt
cagggtcagt gactgtctgg tttctctgag gtgaggctgg 2880aatataggtc
accttgaaga ctaaagaggg gtccaggggc ttctgcacag gcagggaaca
2940gaatgtggaa caatgacttg aatggttgat tcttgtgtga caccaggaat
tggcataatg 3000tctgagttgc ccaggggtga ttctagtcag actctggggt
ttttgtcggg tatagaggaa 3060aaatccacta ttgtgattac tatgctatgg
actactgggg tcaaggaacc tcagtcaccg 3120tctcctcagg taagaatggc
ctctccaggt ctttattttt aacctttgtt atggagtttt 3180ctgagcattg
cagactaatc ttggatattt gtccctgagg gagccggctg agagaagttg
3240ggaaataaac tgtctaggga tctcagagcc tttaggacag attatctcca
catctttgaa 3300aaactaagaa tctgtgtgat ggtgttggtg gagtccctgg
atgatgggat agggactttg 3360gaggctcatt tgaagaagat gctaaaacaa
tcctatggct ggagggatag ttggggctgt 3420agttggagat tttcagtttt
tagaataaaa gtattagttg tggaatatac ttcaggacca 3480cctctgtgac
agcatttata cagtatccga tgcataggga caaagagtgg agtggggcac
3540tttctttaga tttgtgagga atgttccgca ctagattgtt taaaacttca
tttgttggaa 3600ggagagctgt cttagtgatt gagtcaaggg agaaaggcat
ctagcctcgg tctcaaaagg 3660gtagttgctg tctagagagg tctggtggag
cctgcaaaag tccagctttc aaaggaacac 3720agaagtatgt gtatggaata
ttagaagatg ttgcttttac tcttaagttg gttcctagga 3780aaaatagtta
aatactgtga ctttaaaatg tgagagggtt ttcaagtact cattttttta
3840aatgtccaaa attcttgtca atcagtttga ggtcttgttt gtgtagaact
gatattactt 3900aaagtttaac cgaggaatgg gagtgaggct ctctcataac
ctattcagaa ctgactttta 3960acaataataa attaagtttc aaatattttt
aaatgaattg agcaatgttg agttggagtc 4020aagatggccg atcagaacca
gaacacctgc agcagctggc aggaagcagg tcatgtggca 4080aggctatttg
gggaagggaa aataaaacca ctaggtaaac ttgtagctgt ggtttgaaga
4140agtggttttg aaacactctg tccagcccca ccaaaccgaa agtccaggct
gagcaaaaca 4200ccacctgggt aatttgcatt tctaaaataa gttgaggatt
cagccgaaac tggagaggtc 4260ctcttttaac ttattgagtt caacctttta
attttagctt gagtagttct agtttcccca 4320aacttaagtt tatcgacttc
taaaatgtat ttagaattca ttttcaaaat taggttatgt 4380aagaaattga
aggactttag tgtctttaat ttctaatata tttagaaaac ttcttaaaat
4440tactctatta ttcttccctc tgattattgg tctccattca attcttttcc
aatacccgaa 4500gcatttacag tgactttgtt catgatcttt tttagttgtt
tgttttgcct tactattaag 4560actttgacat tctggtcaaa acggcttcac
aaatcttttt caagaccact ttctgagtat 4620tcattttagg agaaagactt
tttttttaaa tgaatgcaat tatctagact tatttcagtt 4680gaacatgctg
gttggtggtt gagaggacac tcagtcagtc agtgacgtga agggcttcta
4740agccagtcca catgctctgt gtgaactccc tctggccctg cttattgttg
aatgggccaa 4800aggtctgaga ccaggctgct gctgggtagg cctggacttt
gggtctccca cccagacctg 4860ggaatgtatg gttgtggctt ctgccaccca
tccacctggc tgctcatgga ccagccagcc 4920tcggtggctt tgaaggaaca
attccacaca aagactctgg acctctccga aaccaggcac 4980cgcaaatggt
aagccagagg cagccacagc tgtggctgct gctcttaaag cttgtaaact
5040gtttctgctt aagagggact gagtcttcag tcattgcttt agggggagaa
agagacattt 5100gtgtgtcttt tgagtaccgt tgtctgggtc actcacattt
aactttcctt gaaaaactag 5160t 516184932DNAMus musculus 8tagcagggtg
tagagggatc tcctgtctga caggaggcaa gaagacagat tcttacccct 60ccatttctct
tttatccctc tctggtcctc agagagtcag tccttcccaa atgtcttccc
120cctcgtctcc tgcgagagcc ccctgtctga taagaatctg gtggccatgg
gctgcctggc 180ccgggacttc ctgcccagca ccatttcctt cacctggaac
taccagaaca acactgaagt 240catccagggt atcagaacct tcccaacact
gaggacaggg ggcaagtacc tagccacctc 300gcaggtgttg ctgtctccca
agagcatcct tgaaggttca gatgaatacc tggtatgcaa 360aatccactac
ggaggcaaaa acaaagatct gcatgtgccc attccaggta agaaccaaac
420cctcccagca ggggtgccca ggcccaggca tggcccagag ggagcagcgg
ggtggggctt 480aggccaagct gagctcacac cttgaccttt cattccagct
gtcgcagaga tgaaccccaa 540tgtaaatgtg ttcgtcccac cacgggatgg
cttctctggc cctgcaccac gcaagtctaa 600actcatctgc gaggccacga
acttcactcc aaaaccgatc acagtatcct ggctaaagga 660tgggaagctc
gtggaatctg gcttcaccac agatccggtg accatcgaga acaaaggatc
720cacaccccaa acctacaagg tcataagcac acttaccatc tctgaaatcg
actggctgaa 780cctgaatgtg tacacctgcc gtgtggatca caggggtctc
accttcttga agaacgtgtc 840ctccacatgt gctgccagtg agtggcctgg
gctaagccca atgcctagcc ctcccagatt 900agggaagtcc tcctacaatt
atggccaatg ccacccagac atggtcattt gctccttgaa 960ctttggctcc
ccagagtggc caaggacaag aatgagcaat aggcagtaga ggggtgagaa
1020tcagctggaa ggaccagcat cttcccttaa gtaggtttgg gggatggaga
ctaagctttt 1080ttccaacttc acaactagat atgtcataac ctgacacagt
gttctcttga ctgcaggtcc 1140ctccacagac atcctaacct tcaccatccc
cccctccttt gccgacatct tcctcagcaa 1200gtccgctaac ctgacctgtc
tggtctcaaa cctggcaacc tatgaaaccc tgaatatctc 1260ctgggcttct
caaagtggtg aaccactgga aaccaaaatt aaaatcatgg aaagccatcc
1320caatggcacc ttcagtgcta agggtgtggc tagtgtttgt gtggaagact
ggaataacag 1380gaaggaattt gtgtgtactg tgactcacag ggatctgcct
tcaccacaga agaaattcat 1440ctcaaaaccc aatggtaggt atcccccctt
cccttcccct ccaattgcag gacccttcct 1500gtacctcata gggagggcag
gtcctcttcc accctatcct cactactgtc ttcatttaca 1560gaggtgcaca
aacatccacc tgctgtgtac ctgctgccac cagctcgtga gcaactgaac
1620ctgagggagt cagccacagt cacctgcctg gtgaagggct tctctcctgc
agacatcagt 1680gtgcagtggc ttcagagagg gcaactcttg ccccaagaga
agtatgtgac cagtgccccg 1740atgccagagc ctggggcccc aggcttctac
tttacccaca gcatcctgac tgtgacagag 1800gaggaatgga actccggaga
gacctatacc tgtgttgtag gccacgaggc cctgccacac 1860ctggtgaccg
agaggaccgt ggacaagtcc actggtaaac ccacactgta caatgtctcc
1920ctgatcatgt ctgacacagg cggcacctgc tattgaccat gctagcgctc
aaccaggcag 1980gccctgggtg tccagttgct ctgtgtatgc aaactaacca
tgtcagagtg agatgttgca 2040ttttataaaa attagaaata aaaaaaatcc
attcaaacgt cactggtttt gattatacaa 2100tgctcatgcc tgctgagaca
gttgtgtttt gcttgctctg cacacaccct gcatacttgc 2160ctccaccctg
gcccttcctc taccttgcca gtttcctcct tgtgtgtgaa ctcagtcagg
2220cttacaacag acagagtatg aacatgcgat tcctccagct acttctagat
atatggctga 2280aagcttgcct aacctggtgc aggcagcatt caggcacata
tatagacaca catgcattta 2340tacatagata tataggtaca catgtgtaga
cacatacatg aatgtgtatt catggacaca 2400cagacaaagg tacacatata
tacacatgag ttcatgcgca cacacatgca tggacactta 2460caaacgcctt
cagagacaaa taggcataga cacacaacca ctcacagaaa cagataccaa
2520tatgcatggt cctgtgtaca cagaaacaga ctataggcaa atatacacaa
ataaactata 2580tagatacaaa gatatgcata tacacacatg tacagaaaca
tcttcacatg tgtacactaa 2640catgtgaaca ggtatagcac acagatacac
ctggactctg accagggctg taatctccaa 2700ggctcacggc tcagagagcc
tacactaggc tgggtcactg atactcctca ggagcccact 2760ctatgattgg
gagagataac cccaggtaca aagtatgcct atctgtctca acaccatggg
2820gcagaagata ctccactaac cacccatgac agaaagttag ccttggctgt
gtctccatta 2880atagaacacc tcagaagacc aatgtgaaat tgcctaaccc
actcacaccc accctgatct 2940ccagttcaaa atgcagaaaa cataatgcag
ttgtccaaaa gatgccccaa ccacacacac 3000acacacacac acacacacac
acacacacac acacacacac acacacacac accatcaagg 3060agcctctgta
aggagtcacc acccaataac actgcctctt tgggctcata tcctggacat
3120tcttcatatt catatccatt tggggcctag gctttagata tccccaaggg
ctcatcttta 3180cagggatcag agatcccaat aaatgccctg gtcccacagc
ctccctcagg tatctgtctg 3240tttatctctt ggtaccaaga cccaacattg
ctggcagggg taggacaagc aacgcacggg 3300aactctgatc aaagaaagtc
atgagatgcc tgagtccttc aggaagtaag gagggacaac 3360ctctggtatc
cctgttctta ttgctaaagc ccaagagaca gggagacctg ctctaaattc
3420tcagtctaaa cagcaccgat ggcaccacct gctcagggaa agtccagagc
acaccaatat 3480cattttgcca cagttcctga gtctgccttt acccaggtcc
atacattgca tctgtcttgc 3540ttgctctgct gccccagggc tcctggaaca
aaggctccaa attagtgtgt cctacagctt 3600ggcctgttct gtgcctccgt
ctagcttgag ctattagggg accagtcaat actcgctaag 3660attctccaga
accatcaggg caccccaacc cttatgcaaa tgctcagtca ccccaagact
3720tggcttgacc ctccctctct gtgtcccttc atagaggggg aggtgaatgc
tgaggaggaa 3780ggctttgaga acctgtggac cactgcctcc accttcatcg
tcctcttcct cctgagcctc 3840ttctacagca ccaccgtcac cctgttcaag
gtagtgtggt tgtggggctg aggacacagg 3900gctgggacag ggagtcacca
gtcctcactg cctctacctc tactccctac aagtggacag 3960caattcacac
tgtctctgtc acctgcaggt gaaatgactc tcagcatgga aggacagcag
4020agaccaagag atcctcccac agggacacta cctctgggcc tgggatacct
gactgtatga 4080ctagtaaact tattcttacg tctttcctgt gttgccctcc
agcttttatc tctgagatgg 4140tcttctttct agactgacca aagacttttt
gtcaacttgt acaatctgaa gcaatgtctg 4200gcccacagac agctgagctg
taaacaaatg tcacatggaa ataaatactt tatcttgtga 4260actcacttta
ttgtgaagga atttgttttg tttttcaaac ctttcctgcg gtgttgacag
4320cccaaggatt atctgaatag agcttaggaa ctggaaatgg aacagtgcag
tctgatggta 4380cttaagggag aaagagggaa aggaggtgtg gaagaagaaa
aaagagaagc agagggggag 4440gggagaaggg agagggagag ggagagggag
agggagaggg agagggagag ggagagagag 4500agagagagag agagagagag
agagagagag agagagcatg cactctaaca gcaaagtaca 4560acacaggcag
ccaatggata gcactctggt tatctaccct gatggaagaa gggaagtagg
4620gcagagaaaa ttccaggcct aatctcccaa aagcaacaga acctggaaac
tagcctctag 4680ccttaggtct ctgctctgtc cccagcccac catcttgggc
tggtgttgct tcaagctagt 4740aatttaggtc ttatcccaaa gctttgtggt
atgtgggtgt gcctttgggg agttggctga 4800gattttgaag atgtttgtac
ctctcccaca acatgacaag ccctaggggt tagtcaataa 4860ctcaaattct
ctgtctatga caactgctgt atgactatat gaagaaatgg gataaagatg
4920ctatagtcac tc 4932
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